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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Experiment start date (Animal Arrival) - 07 April 2015 to Completion date of experimental phase (Foetal pathology): 23 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium Tungstate
Target: Fused tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
ICH Harmonised Tripartite Guideline: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2): finalised (Step 4) November 2005.
Deviations:
no
Principles of method if other than guideline:
ICH Harmonised Tripartite Guideline: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2): finalised (Step 4) November 2005.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2214
- Expiration date of the lot/batch: 01 March 2017
- Purity test date: 85.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item will be stored at room temperature, protected from light.
- Stability under test conditions: Test item formulations at concentrations of 0.5 mg/mL to 50 mg/mL in vehicle have been shown to be stable for up to 14 days at room temperature, when stored refrigerated and when frozen (approximately -80 ºC) (Kymos Reference S15/341-KE). On this basis, formulations, including Control, were stored refrigerated prior to use.

Test animals

Species:
rat
Strain:
other: Crl:CD (SD
Remarks:
Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: On the first day of dosing they weighed 213 to 274 g.
- Housing: Females were housed individually in grid-floor cages over paper lined trays.
- Diet and water (e.g. ad libitum): A pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England, and mains tap water (in bottles) will be freely available.
- Acclimation period: The animals were acclimatised within the study room for at least two days after arrival. Towards the end of this period the animals were re-examined to confirm their suitability for use.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target ranges 19 °C to 23 °C
- Humidity (%): Target ranges 40 % to 70 %,
- Air changes (per hr): Room was air-conditioned
- Photoperiod (hrs dark / hrs light): The room was illuminated by fluorescent light set to give a cycle of 12 hours light and 12 hours dark.

IN-LIFE DATES: From: To: 07 April 2015-23 April 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated for dosing as a solution in the vehicle (purified water). Separate formulations were prepared for each dose level. The weighed quantity of test item was mixed in the appropriate quantity of vehicle. The formulations were prepared within the known stability period (14 days).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations for use on the first day and towards the end of dosing was analysed to determine their achieved concentrations. Vehicle (for Controls) was also be analysed on these occasions to confirm the absence of the test item. Analysis was conducted at Kymos Pharma Services using a validated method (code C003-MP0023).
Details on mating procedure:
The females were obtained from the supplier timed-mated and were by Day 1 to Day 4 of gestation on arrival. For mating, each female were paired with a sexually mature male of the same strain. The day on which mating is detected was designated Day 0 of gestation.
Duration of treatment / exposure:
Animals wiere dosed once daily, from Day 6 of gestation to Day 17 of gestation inclusive. Control animals received the vehicle only, following the same regimen as the other groups.
Frequency of treatment:
Animals were dosed once daily
Duration of test:
From Day 6 of gestation to Day 17 of gestation inclusive
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females per group per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels have been selected after examining existing toxicity data and on the basis of results from a preliminary dose range finding study (Sequani Study Number: OHS0004).
- Rationale for animal assignment (if not random): Allocation to groups wiere performed using a stratified randomisation procedure based on body weight ranges recorded on arrival.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Animals will be examined twice daily for mortality and morbidity. During the treatment period each animal was routinely checked pre-dose and soon after completion of dosing. On week days, additional observations were made approximately 1 hour after dosing and approximately 4 hours after dosing or at the end of the working day (whichever is sooner).

DETAILED CLINICAL OBSERVATIONS: Yes
All animals will be examined daily for clinical signs of toxicity or changes in behaviour and appearance from the start of treatment.

BODY WEIGHT: Yes
Day 0 of gestation body weight will be recorded by the supplier. At Sequani, body weights will be recorded daily from Day 5 to 18 of gestation, inclusive, then again on Day 20 of gestation.

FOOD CONSUMPTION: Yes
The amount of food consumed by each animal will be recorded over Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, and 18 to 20 of gestation.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: Females were killed on Day 20 of gestation
- Organs examined: The animals were weighed, the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Gravid uterus and placenta weights were recorded and organs or tissues showing any macroscopic abnormalities were removed and retained in fixative

Ovaries and uterine content:
For pregnant females the following observations will be made: 1. Number of corpora lutea 2. Number and distribution of implantations in each uterine horn, classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses 3. Gravid uterus weight 4. Placental weight
The implantations are numbered separately for the right and left horns. Numbering is sequential, commencing at the ovarian end through to the cervix. The live foetuses and their placentae will be removed and the uterus and ovaries will be retained in neutral buffered formaldehyde. Gravid uterus and placenta weights will be recorded for pregnant females killed on Day 20 of gestation only.
Fetal examinations:
Live foetuses will be killed by rapid cooling followed by immersion in fixative. The following observations will be made for live foetuses killed on Day 20 of gestation: 1. Foetal weights; 2. Foetal sexes, and 3. Foetal abnormalities (to include external, fresh visceral, fixed visceral and/or skeletal examinations)
Statistics:
Comparisons: Group 1 against Groups 2, 3 and 4.

Statistical tests and parameters: Data was processed to give group mean values and standard deviations, where appropriate. Where the data allow, the following methods wiere used for statistical analysis. Depending on the nature of the data set to be analysed, appropriate tests were applied. Where parametric tests may be appropriate they preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions fails a log transformation was applied before retesting. If the transformation fails, appropriate non-parametric tests was applied.

Proportions of foetuses affected were treated as continuous nonparametric data, using one-sided step-wise Jonckheere Tests. Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 also were noted.
Indices:
1) Pre-implantation loss (%) = [(no. corpora lutea – no. implantation sites)/ number of corpora lutea] x 100
2) Post-implantation loss (%) = [(no. implantation sites – no. live foetuses)/no. of implantation sites] x 100

Mean foetal body weights will be calculated separately by sex for each litter; group mean body weights will be calculated (separately by sex) from the litter means. The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages was calculated from the litter percentages. The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Most animals given 250 mg/kg/day had decreased activity by four hours after dosing on Day 6 of gestation, however, this was an isolated occasion. Treatment-related adverse clinical signs, with decreases in activity seen between one and four hours after dosing on Days 6 and/or 7 of gestation considered to be transient.
Mortality:
mortality observed, treatment-related
Description (incidence):
Female 28 (250 mg/kg/day) was killed on Day 9 of gestation due to marked body weight loss (-14 %) from the start of dosing; this animal also ate notably less than Controls (-51 %). There were no deaths at 50 or 100 mg/kg/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Female 28 (250 mg/kg/day) was killed on Day 9 of gestation due to marked body weight loss (-14 %) from the start of dosing; this animal also ate notably less than Controls (-51 %). For the remaining animals in this group, although food intake was comparable with Controls, marked body weight losses were also apparent from Day 8 of gestation (up to 12 %), such that by Day 11 of gestation, this dose level was considered unsuitable for future use and the remaining animals in this group killed. There was no effect of sodium tungstate on body weight gain at 50 or 100 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no effect of sodium tungstate on body weight gain or food intake at 50 or 100 mg/kg/day.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Female 28 (250 mg/kg/day) was killed on Day 9 and macroscopic necropsy identified a pale corticomedullary junction in each kidney. At necropsy, all animals, presented no macroscopic abnormalities in animals exposed to 50 or 100 mg/kg/day.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
At 50 or 100 mg/kg/day, there were no effects on the uterine/implantation data. At 100 mg/kg/day the incidence of pre- and post-implantation losses were similar to Controls.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
In the group given 100 mg/kg/day, mean number of live foetuses was lower than Controls, however, this reflected a lower mean number of corpora lutea as the incidence of pre- and post- implantation losses were similar to Controls; therefore, this difference was unrelated to sodium tungstate.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At necropsy, all animals, except Female 26, were pregnant. All females in the groups given 0, 50 or 100 mg/kg/day were pregnant.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No changes on group mean values of Litter weights (g) / foetal data
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
In the group given 100 mg/kg/day, mean number of live foetuses was lower than Controls, however, this reflected a lower mean number of corpora lutea as the incidence of pre- and post-implantation losses were similar to Controls; therefore, this difference was unrelated to sodium tungstate
Changes in sex ratio:
no effects observed
Description (incidence and severity):
At 50 or 100 mg/kg/day there was no adverse effect on sex ratio.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean foetal weight was marginally higher than Controls in the group given 100 mg/kg/day, but was attributed to the smaller litter size and not an effect of sodium tungstate.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
Maternal treatment at 50 or 100 mg/kg/day was not associated with any external foetal abnormalities, with only one foetus in the group given 100 mg/kg/day having the minor abnormality of a filamentous tail tip. As no other foetuses had this or similar abnormalities, this abnormality was considered unrelated to Sodium Tungstate
Skeletal malformations:
not specified
Visceral malformations:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Administration of 50 or 100 mg/kg/day sodium tungstate, once daily, by oral gavage to the rat, from Day 6 to Day 17 of gestation, was well tolerated, with no maternal or embryo-foetal toxicity. Administration of 250 mg/kg/day sodium tungstate elicited marked maternal toxicity which necessitated the termination of the group by Day 11 of gestation. Consequently, a dose level below 250 mg/kg/day is considered a suitable high dose level for the subsequent pivotal embryo-foetal development study.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for fused tungsten carbide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which will be used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.